计算单位体积质粒的拷贝数（copy number）

08/19/2022 22:34 • 实验方法 • 阅读 987

1. 第一步，计算质粒的分子量。假设一个DNA碱基对（bp）的平均重量为650道尔顿（Da），一摩尔的碱基对（bp）的重量为650克，即任何双链DNA模板的分子量可以通过取其长度（bp）与每个碱基对重量的乘积来估计。
  First, calculate the molecular weight of the plasmid. Assuming that the average weight of a DNA base pair (bp) is 650 Daltons (Da) and that one mole of a bp weighs 650 g, the molecular weight of any double-stranded DNA template can be estimated by taking the product of its length (in bp) and weight per base pair.
  计算公式：Plasmid molecular weight [g/mol] = plasmid size [bp] x 650 [Da/bp]
2. 计算每微升中质粒的摩尔数。
  Next, calculate the number of moles of plasmid per microliter.
  计算公式：Moles of plasmid per microliter = plasmid concentration [g/µL] / plasmid molecular weight [g/mol]
3. 然后，用阿伏伽德罗数（6.022 x 10²³molecules/mole）计算每微升的质粒分子数。
  Then, calculate the number of plasmid molecules per microliter, using Avogadro’s number (6.022 x 10²³molecules/mole).
  计算公式：Molecules of plasmid per microliter = moles of plasmid per microliter [moles/µL] x Avogadro’s number [molecules/mole]
4. 稀释质粒原液（分子/微升），得到100微升的溶液，所需浓度为1 x 109个分子（（vg）每微升）。
  Finally, dilute the plasmid stock (molecules/µL) to obtain a 100 µL solution with the desired concentration of 1 x 10⁹molecules (vector genomes (vg) per microliter).
  计算公式：Plasmid stock (100 µL) = (desired concentration [molecules/µL] x 100 µL) / plasmid molecules [molecules/µL]